Tissue transglutaminase expression promotes cell attachment, invasion and survival in breast cancer cells

Distant metastasis is frequently observed in patients with breast cancer and is a major cause of cancer-related deaths in these patients. Currently, very little is known about the mechanisms that underlie the development of the metastatic phenotype in breast cancer cells. We previously found that metastatic breast cancer cells express high levels of tissue transglutaminase (TG2), but established no direct link between TG2 and metastasis. In this study, we hypothesized that TG2 plays a role in conferring the metastatic phenotype to breast cancer cells. The results obtained suggested that increased expression of TG2 in breast cancer cells contributes to their increased survival, invasion and motility. We further found that TG2 protein in a metastatic breast cancer MDA-MB231 cells was present on the cell surface in close association with integrins beta1, beta4 and beta5. Downregulation of endogenous TG2 by small interfering RNA inhibited fibronectin (Fn)-mediated cell attachment, survival and invasion. Conversely, ectopic expression of TG2 augmented invasion of breast cancer cells and attachment to Fn-coated surfaces. We conclude that TG2 expression in breast cancer cells plays an important role in the development of the metastatic phenotype.     

Up-regulation of alpha 2 beta 1 integrin cell-surface expression protects A431 cells from epidermal growth factor-induced apoptosis

High epidermal growth factor (EGF) concentration (10(-8) M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell-cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell-cell survival signals probably mediated by calcium-requiring molecules such as integrins. From a panel of integrins tested, only alpha 2 beta 1 integrin cell-surface expression was up-regulated after high apoptotic EGF treatment, and this up-regulation was not observed under a growth-stimulatory EGF concentration (10(-11) M). Double-labeling analysis (alpha 2 beta 1/DNA) implicated alpha 2 beta 1 integrin in the resistance process since 99% of cells that up-regulated alpha 2 beta 1 integrin survived a high dose of EGF. Moreover, the involvement of alpha 2 beta 1 integrin up-regulation in the survival of A431 cells that escape EGF-induced apoptosis was verified using the blocking anti-alpha 2 beta 1 integrin antibody, which was shown to decrease the survival of EGF-stimulated cells. Furthermore, under our culture conditions, alpha 2 beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions, suggesting that alpha 2 beta 1 integrin is involved more directly in cell-cell interaction than in cell-substrate adhesion. Our results provide evidence that EGF-induced up-regulation of alpha 2 beta 1 integrin contributes to the enhancement of cell-cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF-induced death by alpha 2 beta 1 integrin signaling.     

Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression

Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.     

Alterations in integrin expression modulates invasion of pancreatic cancer cells

Background

Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood.

Methods

In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression.

Results

Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin β1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion.

Conclusion

Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.     

CARMA3 is overexpressed in human glioma and promotes cell invasion through MMP9 regulation in A172 cell line

Caspase recruitment domain-containing membrane-associated guanylate kinase protein 10 or CARMA3 (CARD10) is a recently characterized oncoprotein involved in the progression of several human malignancies. The present study aims to investigate the expression pattern and biological roles of CARMA3 protein in human glioma. CARMA3 expression was analyzed in 97 glioma specimens using immunohistochemistry. We observed negative staining in normal astrocytes and positive staining of CARMA3 in 25 out of 97 (25.8 %) glioma samples. Overexpression of CARMA3 correlated with tumor grade (p?<?0.001). Small interfering RNA knockdown was performed in A172 cell line with relatively high CARMA3 expression. Using colony formation assay and Matrigel invasion assay, we showed that CARMA3 depletion in A172 cell line inhibited cell proliferation and cell invasion. In addition, mRNA and protein levels of matrix metallopeptidase 9 (MMP9) were downregulated, indicating CARMA3 might regulate invasion through MMP9. In conclusion, CARMA3 serves as an oncoprotein in human glioma by regulating cell invasion, possibly through MMP9 regulation.     

Up-regulation of SIBLING proteins and correlation with cognate MMP expression in oral cancer

Various combinations of the SIBLING family of proteins have been found to be up-regulated in many human cancers and have been linked to different stages of tumor progression, including metastasis. Bone sialoprotein (BSP), osteopontin (OPN) and dentin matrix protein 1 (DMP1) specifically bind and activate MMP-2, MMP-3, and MMP-9, respectively. These proteases have also been shown to play important roles in oral squamous cell carcinoma (OSCC) invasion and metastasis. However, with the exception of OPN, there are no reports on the expression of the family of five SIBLING proteins in OSCC. This study examines the expression patterns of the SIBLING family (and MMP partners when known) in OSCC, correlating expression to outcome variables. Archived paraffin sections of 87 cases of primary OSCC were screened by immunohistochemistry for the SIBLINGs and their MMP partners. Three SIBLINGs (BSP, DSPP, and OPN), were expressed in OSCC, while DMP1 and MEPE expression were never observed. Furthermore, BSP and OPN were always expressed with their known MMP partners, MMP-2 and MMP-3, respectively. Poorly differentiated tumors exhibited reduced or no immunoreactivity for BSP and OPN but increased immunoreactivity for DSPP. Seventy eight (90%) cases were positive for BSP and DSPP, while 79 cases (91%) were positive for OPN. Overall, 91% of the cases were positive for at least one SIBLING. There were no correlations between SIBLING expression and tumor size ("T"; of the Union Internationale Contre le Cancer [UICC]-TNM classification for OSCC), and between SIBLING expression and lymph node spread for the T1/T2 tumors. The levels of DSPP expression for floor of mouth and retromolar region tumors were higher than for tongue tumors. Statistically significant correlations were, however, found between the expression levels of BSP and MMP-2 (p<0.0001), BSP and MMP-3 (p<0.0001), and OPN and MMP-3 (p<0.0024). We conclude that BSP, DSPP, and OPN are highly up-regulated in OSCC. While the production of these SIBLINGs is independent of T, they correlate with oral location of tumor, cognate MMP expression, and for DSPP, the degree of tumor differentiation.     

Unique expression of integrin fibronectin receptors in human neuroblastoma cell lines.

Cultured human neuroblastoma cells can be classified morphologically into 3 types: neuroblastic (N), intermediate (I) and substrate adherent (S). Neuroblastoma cells of all types were found to attach and display distinct morphological characteristics on fibronectin, with S-type cells attaching better than N-type cells. Studies of the expression of integrin fibronectin receptors (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1 and alpha V beta 1) were carried out using a total of 26 morphologically distinct cell lines and their subpopulations. Fluorescence-activated cell sorting (FACS) analysis and immunoprecipitation revealed that all S-type cells expressed abundant alpha 5 beta 1, while N-type cells barely expressed this molecule. Although alpha 3 beta 1 expression of S-type cells was also higher than that of N-type cells, some N-type cells had significantly increased levels of this molecule. alpha 4 beta 1 was found to be randomly expressed. All cell lines tested expressed alpha V beta 1. Human neuroblastoma cells, the majority of which are N-type cells with very low alpha 5 beta 1 expression, are also contrasted with other childhood cancer cells (rhabdomyosarcoma, Ewing's sarcoma, and glioma), all of which expressed high levels of alpha 5 beta 1. The characteristic expression of integrin fibronectin receptors may account for the clinically unique tumor behavior, and the immunohistochemical staining for integrins may become a useful alternative to conventional histology in differential diagnosis and a marker for prognosis in neuroblastoma.     

Runx2通过促进MMP9的表达增强肝细胞肝癌迁移侵袭能力

  目的   研究Runx2对肝癌细胞中MMP9的表达以及其对肝癌细胞侵袭迁移能力的影响。  方法   通过免疫组织化学染色法对2005年12月至2015年12月天津医科大学肿瘤医院和天津医科大学总医院189例肝细胞肝癌病例标本中Runx2和MMP9的表达进行分析;将Runx2过表达质粒和干扰质粒分别转染至肝癌细胞系HepG2和SMMC7721细胞中。使用Western blot法检测转染后HepG2和SMMC7721细胞中Runx2和MMP9的表达情况;划痕、侵袭实验检测Runx2对肝癌细胞迁移和侵袭能力的影响。  结果   免疫组织化学结果显示Runx2的表达和MMP9的表达相关。在HepG2细胞中上调Runx2的表达,促进了MMP9的表达,增强了HepG2细胞迁移侵袭能力;在SMMC7721细胞中下调Runx2的表达,降低了MMP9的表达,抑制了SMMC7721细胞的迁移侵袭能力。  结论   Runx2的表达可能参与促进肝细胞肝癌细胞MMP9的表达,进而促进肝细胞肝癌的迁移侵袭能力。      

CXCR4/SDF1 mediate hypoxia induced chondrosarcoma cell invasion through ERK signaling and increased MMP1 expression

Background  

Chondrosarcoma is a disease that does not respond to conventional cytotoxic chemotherapy and expression of MMP1 is a marker for a poor prognosis. The mechanism of increased MMP1 expression in chondrosarcoma is not completely known. Our goal is to identify molecular pathways that could serve as therapeutic targets. Chondrosarcoma become hypoxic as they grow, are capable of eliciting an angiogenic response, and typically metastasize to the lungs. The present study determined the effect of hypoxia and specifically HIF-1a on expression of CXCR4 and MMP1 and their role in chondrosarcoma cell invasion.     

Lysophosphatidic acid promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells

Ovarian cancer is an highly metastatic disease characterized by ascites formation and diffuse i.p. adhesion, invasion, and metastasis. Levels of lysophosphatidic acid (LPA) are elevated in the plasma of patients with ovarian carcinoma, including 90% of patients with stage I disease, suggesting that LPA may promote early events in ovarian carcinoma dissemination. Expression of matrix metalloproteinases (MMPs) is also up-regulated in ovarian cancer tissues and ascites, and numerous studies have provided evidence for a direct role of MMPs in i.p. invasion and metastasis. Using three-dimensional type I collagen cultures or immobilized beta1 integrin subunit-specific antibodies, we previously demonstrated that beta1 integrin clustering promotes activation of proMMP-2 and processing of membrane type 1 MMP in ovarian cancer cells (S. M. Ellerbroek et al., Cancer Res., 59: 1635-1641, 1999). In the current study, the effect of LPA on MMP expression and invasive activity was investigated. Treatment of ovarian cancer cells with pathophysiological levels of LPA increased cellular adhesion to type I collagen and beta1 integrin expression. A significant up-regulation of MMP-dependent proMMP-2 activation was observed in LPA-treated cells, leading to enhanced pericellular MMP activity. As a result of increased MMP activity, haptotactic and chemotactic motility, in vitro wound closure, and invasion of a synthetic basement membrane were enhanced. These data indicate that LPA contributes to metastatic dissemination of ovarian cancer cells via up-regulation of MMP activity and subsequent downstream changes in MMP-dependent migratory and invasive behavior.     

The metastasis-associated molecule C4.4A promotes tissue invasion and anchorage independence by associating with the alpha6beta4 integrin

C4.4A is a metastasis-associated molecule that functions appear to rely on associated alph6beta4 integrin. To corroborate the impact of the C4.4A-alpha6beta4 integrin association on metastasis formation, C4.4A was knocked-down in a highly metastatic rat pancreatic adenocarcinoma (ASML, ASML-C4.4Akd).Metastasis formation by ASML-C4.4Akd cells after intrafootpad application was strongly retarded in draining nodes and lung colonization was rare. Furthermore, cisplatin treatment significantly prolonged the survival time only of ASML-C4.4Akd-bearing rats. ASML-C4.4Akd cells display reduced migratory activity and impaired matrix protein degradation due to inefficient MMP14 activation; loss of drug-resistance is due to mitigated PI3K/Akt pathway activation. These losses of function rely on the laminin receptor C4.4A recruiting activated alpha6beta4 integrin into rafts, where C4.4A cooperates with alpha6beta4 and via alpha6beta4 with MMP14. Within this raft-located complex, MMP14 provokes focalized matrix degradation and mostly alpha6beta4 integrin promotes BAD phosphorylation and upregulated Bcl2 and BclXl expression.Thus, metastasis-promoting activities of C4.4A are not genuine characteristics of C4.4A. Instead, the raft-located laminin receptor C4.4A recruits alpha6beta4 integrin and supports via the alpha6beta4 integrin MMP14 activation. Thereby C4.4A acts as a linker to facilitate several steps in the metastatic cascade. Taking the restricted C4.4A expression in non-transformed tissue, this knowledge should pave the way toward the use of C4.4A as a therapeutic target.     

Implications of increased tissue transglutaminase (TG2) expression in drug-resistant breast cancer (MCF-7) cells

The development of resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of breast cancer. Ways to block or overcome this resistance are objects of intense research. We have previously shown that cancer cells selected for resistance against chemotherapeutic drugs or isolated from metastatic tumor sites have high levels of a calcium-dependent protein crosslinking enzyme, tissue transglutaminase (TG2) but no direct link between TG2 and resistance was established. As TG2 can associate with the beta members of the integrin family of proteins, we hypothesized that TG2 promotes cell survival signaling pathways by activating integrins on the surface of these cells. To test this hypothesis, we studied the expression of TG2 and its interaction with various integrins in drug-resistant MCF-7 breast cancer cells. TG2 closely associated with beta1 and beta5 integrins on the surface of drug-resistant MCF-7 (MCF-7/Dox and MCF-7/RT) cells. The incubation of TG2-expressing drug-resistant MCF-7 cells on fibronectin (Fn)-coated surfaces strongly activated focal adhesion kinase, an event that leads to the activation of several downstream signaling pathways and, in turn, can confer apoptosis-resistant phenotype to cancer cells. The role of TG2 in Fn-mediated cell attachment, cell growth, and cell survival functions was further analysed by small interfering RNA (siRNA) approach. Inhibition of TG2 by siRNA-inhibited Fn-mediated cell attachment and cell survival functions in drug-resistant MCF-7 cells. We conclude that the expression of TG2 in breast cancer cells contributes to the development of the drug-resistance phenotype by promoting interaction between integrins and Fn.     

miR-4712-5p在外阴鳞癌A431细胞增殖、侵袭中的作用

目的:筛选外阴鳞状细胞癌差异表达基因,观察其在外阴鳞癌中的作用,探讨其在外阴鳞癌组织、细胞中的作用靶点。方法:采用生物信息学方法筛选差异表达的基因miRNA-4712-5p。其表达在外阴鳞癌临床组织（VS组织）和外阴鳞癌细胞系A431（VS细胞）中通过qRT-PCR方法得到验证。构建过表达模拟物并转染A431细胞后,通过CCK-8实验和Transwell实验判断miRNA-4712-5p在外阴鳞癌细胞中增殖、侵袭和转移的作用,并分析miRNA-4712-5p作用靶点PTEN,Western blot实验和免疫组化实验检测PTEN表达水平。结果:miR-4712-5p在外阴鳞癌组织和细胞中的表达水平显著升高,并可促进外阴鳞癌细胞的增殖和侵袭。生物信息学分析PTEN可能是miR-4712-5p的靶基因。qRT-PCR显示外阴鳞癌组织中PTEN表达显著低于邻近非癌组织。用所构建的miR-4712-5p过表达模拟物转染A431细胞后,PTEN蛋白显著降低。结论:miR-4712-5p在外阴鳞癌组织和细胞中显著增高,可通过降低PTEN蛋白的表达,促进外阴鳞癌细胞的增殖和侵袭。     

Up-regulation of METCAM/MUC18 promotes motility,invasion, and tumorigenesis of human breast cancer cells

Background  

Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells.     

整合素连接激酶在非小细胞肺癌中过度表达及促进肺癌细胞的侵袭和迁移

目的:探讨整合素连接激酶（ILK）在非小细胞肺癌（NSCLC）中的表达及在侵袭和迁移中的作用和相关分子机制。方法:免疫组化法检测ILK蛋白在NSCLC患者中的表达,细胞转染、siRNA干扰、细胞划痕试验、实时定量PCR、Western blot方法探讨ILK在肺癌A549细胞中的表达及分子机制。结果:ILK蛋白在原发性NSCLC组织中过度表达30.6％（33/108）并且和TNM分期（P=0.001）、淋巴结转移（P=0.033）相关。ILK在A549细胞中过度表达并且通过下调E-cadherin,上调波形蛋白、纤维连接蛋白、Snail、Slug导致上皮-间质转化（EMT）。此外,NF-κB抑制剂BAY 11-7028和小干扰靶RNA（siRNA）NF-p65可诱导E-cadherin的表达下调。结论:ILK在原发性NSCLC组织中高表达并与TNM分期和淋巴结转移相关,其促进肺癌细胞的侵袭和迁徙机制可能是经NF-κB信号通路诱导EMT所致。